Journal: PLoS Biology

Article Title: Gut bacteria-derived 5-hydroxyindole is a potent stimulant of intestinal motility via its action on L-type calcium channels

doi: 10.1371/journal.pbio.3001070

Figure Lengend Snippet: (A) Bar graph represents a significantly increased 5-HT release from RIN14B cells after stimulation with 100 μM of 5-HI. Data were analyzed using the 2-tailed unpaired t test (**** p < 0.0001). Error bars represent SEM. ( B) Bar graph and illustrative recording in the rat colon represents the enhanced response of the 5-HI on the gut contractility. ( C) The dose response curve and EC 50 value of 5-HI in the rat colonic tissue. (D–G) Bar graphs and illustrative recordings in the rat colon represent the enhanced response of the 5-HT-induced contractility by ( D) 100 μM 5-HI, which was not inhibited after adding ( E) 1 μM ondansetron, ( F) 1 μM SB-207266, or ( G) 1 μM TTX. ( H–J) Bar graphs and illustrative recordings in the rat colon represent enhanced response of the ACh-induced contractility by ( H) 100 μM 5-HI, which was significantly increased after adding ( I) 100 nM DAU 5884 hydrochloride and was not inhibited after the addition of ( J) 1 μM AQ-RA 741. ( K) Addition of 1 μM nifedipine significantly inhibited 5-HI-induced response. (L) Illustrative recording in the rat colon represents enhanced response by 100 μM 5-HI after colonic tissue contractile apparatus is altered by nifedipine. Data represent 3–9 biological replicates. Data were analyzed using the Wilcoxon matched-pairs (before/after) signed rank test (* p < 0.05; ** p < 0.01). Error bars represent SEM. Quantitative analysis of the organ bath data is described in Materials and method section. The raw data used for quantification of A–K can be found in . 5-HI, 5-hydroxyindole; ACh, acetylcholine; n.s., not significant; TTX, tetrodotoxin.

Article Snippet: To perform the tests with antagonists for the receptors involved in the gut motility, either 1 μM ondansetron (5-HT 3 antagonist; O3639, Sigma), 1 μM SB-207266 (5-HT 4 antagonist; SML1349, Sigma), 1 μM TTX (sodium ion transport blocker on the enteric neurons; TTX; 1078/1, Tocris Bioscience), 1 μM hexamethonium bromide (a nicotinic cholinergic antagonist; 4111, Tocris Bioscience), 1 μM AQ-RA 741 (muscarinic receptor 2 antagonist (M2R); 2292, Tocris Bioscience), 100 nM DAU 5884 hydrochloride (M3R antagonist; 2096; Tocris Bioscience), 1 μM ML204 (TRPC4 antagonist; 4732, Tocris Bioscience), 1 μM SAR 7334 (TRPC6 antagonist; 5831; Tocris Bioscience), 100 μM ATP (A1335; Duchefa Bioscience), or 1 μM nifedipine (L-type Ca 2+ channels antagonist; 1075; Tocris Bioscience) was applied sequentially with either ACh, 5-HT, or 5-HI.

Techniques:

Journal: Science Advances

Article Title: The long noncoding RNA neuroLNC regulates presynaptic activity by interacting with the neurodegeneration-associated protein TDP-43

doi: 10.1126/sciadv.aay2670

Figure Lengend Snippet: ( A ) Relative expression of neuroLNC in the rat central nervous system (CNS) and other tissues, as measured by quantitative real-time PCR (qRT-PCR). Statistical test, one-way ANOVA followed by Bonferroni’s multiple comparison test versus all non-CNS samples (*** P < 0.001). ( B ) RNA fluorescence in situ hybridization (RNA-FISH) of neuroLNC on coronal brain slice of rat (P14). Note the localization to the nuclei of CA1 neurons in the zoom. Scale bars, 0.5 mm (left); 50 μm (right). DAPI, 4′,6-diamidino-2-phenylindole. ( C ) Combined RNA-FISH and immunofluorescence in primary mixed neuroglial cultures. Note that β-III tubulin–positive neurons are neuroLNC positive (left, white arrowhead), while cells positive for the glial marker glial fibrillary acidic protein (GFAP) are neuroLNC negative (right, white arrowhead). Scale bars, 10 μm. ( D ) Quantification of Ca 2+ influx in primary hippocampal neurons upon neuroLNC overexpression following a 20-AP field stimulation at different frequencies. Mean values are plotted (± SEM). Insets indicate the difference in fluorescence measured at the maximum peak for each experiment. Statistical test, unpaired two-tailed Student’s t test (* P < 0.05; ** P < 0.01; and *** P < 0.001). See fig. S5 for details. ( E ) Quantification of Ca 2+ influx as in (D), following shRNA-mediated neuroLNC down-regulation with a 600-AP stimulation at 20 Hz. Upon neuroLNC down-regulation, Ca 2+ influx is significantly decreased. ( F ) Physiological (unstimulated) network activity measured in neurons following overexpression or down-regulation of neuroLNC . Overexpression increases spontaneous activity, while down-regulation has the opposite effect. Unpaired two-tailed Student’s t test (* P < 0.05). ( G ) The number of neuroLNC foci following activity modulation of neurons. Blocking the activity of neurons with TTX or AP5 + CNQX has no effect. The number of neuroLNC foci is promoted by increasing neuronal activity by applying bicuculline or 4-AP. In the upper panels, representative images are shown. Scale bar, 5 μm. Normalized averages are shown. One-way ANOVA followed by Bonferroni’s multiple comparison test versus control (** P < 0.01).

Article Snippet: For the pharmacological modulation of neuronal activity, we used 1 μM TTX (Na + Channel blocker; Cayman Chemical, USA), 20 μM bicuculline methobromide (GABA A receptor antagonist; Tocris Bioscience, Cambridge, UK), 100 μM 4-AP (K + channel blocker; Tocris Bioscience, Cambridge, UK), 10 μM CNQX (AMPA/kainate receptor antagonist; Tocris Bioscience, Cambridge, UK), and 50 μM D-AP5 ( N -methyl- d -aspartate receptor antagonist; Tocris Bioscience, Cambridge, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Fluorescence, In Situ Hybridization, Slice Preparation, Immunofluorescence, Marker, Over Expression, Two Tailed Test, shRNA, Activity Assay, Blocking Assay, Control

Journal: Cell Reports

Article Title: Local Resting Ca 2+ Controls the Scale of Astroglial Ca 2+ Signals

doi: 10.1016/j.celrep.2020.02.043

Figure Lengend Snippet:

Article Snippet: In these experiments, neuronal activity was inhibited by adding the sodium channel blocker TTX (1 μM, Tocris Bioscience) to the extracellular solution.

Techniques: Recombinant, Software